METHODS FOR PREVENTION AND TREATMENT OF CARDIOMETABOLIC SYNDROME AND COMPOSITIONS USED THEREIN
20190365666 ยท 2019-12-05
Inventors
- Jayant Deshpande (Charlottetown, CA)
- Abhijit Bhattacharya (Morristown, NJ, US)
- Vijaya Juturu (Morristown, NJ)
Cpc classification
A61K31/047
HUMAN NECESSITIES
G01N2333/47
PHYSICS
A61K31/045
HUMAN NECESSITIES
G01N33/92
PHYSICS
International classification
Abstract
Beta-cryptoxanthin compositions and methods are described for the management of cardiometabolic syndrome and associated risk factors, in a subject, in need thereof. Methods herein are directed to identifying such subject at risk of developing cardiometabolic syndrome and administering beta-cryptoxanthin composition to assess the condition of an organ. Compositions and methods herein can effectively reduce risk factors of cardiometabolic syndrome, such as hyperlipidemia, insulin resistance, obesity, diabetes, atherosclerosis and/or related cardiovascular disorders. Beta-cryptoxanthin compositions and methods herein can reduce body weight, body fat, glucose levels, and free fatty acids, when administered in effective amounts. The compositions and methods herein can also reduce oxidative stress on organs such as the eye and liver and/or reduce inflammatory and/or oxidative markers, when administered to subjects in need thereof.
Claims
1-7. (canceled)
8. A method for prevention and treatment of eye related complications and liver, comprising administering a beta cryptoxanthin composition in an effective amount to a subject fed with high fat diet; and evaluating effect of composition on, oxidative stress markers and inflammatory makers, to assess overall management of eye and liver, wherein the subject is evaluated on oxidative stress markers and inflammatory markers related to eye and liver.
9. The method of claim 8, wherein the beta-cryptoxanthin composition is administered to the subject fed with high fat diet in a daily dose of about 0.1 to 100 mg/kg body weight and evaluated to assess overall management of eye and liver.
10. The method of claim 8, wherein the amount of the beta cryptoxanthin composition is effective for retarding an accumulation of lipofuscin pigment in retina and preventing causes of retinal neovascularization, retinal vein occlusion, and/or neovascularization in peripheral retina.
11. The method of claim 8, wherein the evaluating includes measuring an activity of at least one selected from the group consisting of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and thiobarbituric acid reactive substances (TBARS) to assess liver function.
12. The method of claim 8, wherein the evaluating includes measuring an activity of at least one selected from the group consisting of heme-oxygenase 1 (HO-1), intercellular adhesion molecule 1 (ICAM-1), Inducible nitric oxide synthase (iNOS), nuclear factor kappa light chain enhancer of activated B cells (NFkB), nuclear factor erythroid derived 2-related factor 2 (Nrf-2), and vascular endothelial growth factor (VEGF) to assess eye function.
13. The method of claim 8, wherein beta-cryptoxanthin is obtained from paprika oleoresin.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0076] The methods described herein are comprised of identifying a subject in need thereof, administering beta-cryptoxanthin composition(s) in an effective amount(s), and evaluating the effect for treatment, prevention and/or management of cardiometabolic syndrome and their associated risk factor(s). The methods described herein can improve conditions associated with cardiometabolic syndrome such as body weight, lipid profile, insulin resistance, blood glucose, reduction in oxidative stress, and inflammatory markers, to protect vital organs like the eye and liver, when administered to a subject, such as for example who is habituated for high fat diet.
[0077] Beta-cryptoxanthin for the compositions as described herein may be obtained by natural resources and are safe for administration and thus useful for nutraceutical purposes.
[0078] Cardiometabolic syndrome, also known as syndrome X, increases the risk of developing cardiovascular disease, particularly atherosclerosis, heart failure, dyslipidemia, diabetes, and associated risk factors, which may be caused mainly due to imbalance of calorie intake and energy utilization. One of the most important causes for this is a high fat diet. The syndrome also affects vital body organs such as liver and eye. Therefore it is important to identify methods for treating and preventing it and associated risk factors thereof by administering compositions which are safe for administration and evaluating the effect in subjects in need thereof.
[0079] The terminology subject is commonly used in the specification to refer to an individual or mammal under test, being treated with compositions herein.
[0080] The terminology subject in need thereof can include specific individuals or mammals who are habituated to a diet rich in high fat and refined carbohydrates, thus lacking in fibers. Such subjects are at high risk of developing cardiometabolic syndrome or symptoms for associated risk factors and/or may be suffering from cardiometabolic syndrome, because of developing abdominal obesity.
[0081] Abdominal obesity drives the progression of multiple risk factors directly, through secretion of excess free fatty acids and inflammatory adipokines, and decreased secretion of adiponectin (Desprs J P et al, 1990; Pouliot M C, 1992; Kissebah A H et al, 1989; Carey V J, 1997; Turkoglu C et al, 2003). Significant effects of abdominal obesity can be dyslipidaemia and insulin resistance, which can provide an indirect, though clinically important, link to the genesis and progression of atherosclerosis and cardiometabolic risk. Excess abdominal obesity is accompanied by elevated levels of C-reactive protein (CRP) and free fatty acids (FFAs), as well as decreased levels of adiponectin. Elevated CRP is an indicator of inflammation. Abdominal obesity may be associated with the inflammation cascade, with adipose tissue expressing a number of inflammatory cytokines. Inflammation is now believed to play a role in the development of atherosclerosis and type 2 diabetes. Elevated levels of CRP are considered to be predictive of cardiovascular disease and insulin resistance.
[0082] Elevated FFA levels appear to play a significant role in the cause of insulin resistance. It has been suggested that elevated FFAs and intracellular lipids inhibit the insulin signaling mechanism, leading to decreased glucose transport to muscle. Adiponectin is an adipose tissue-specific circulating protein which is involved in the regulation of lipid and glucose metabolism. Adiponectin has been shown to be reduced in adults with obesity and type 2 diabetes. Such components help to explain why excess abdominal obesity is considered to be a significant risk to cardiovascular and metabolic health.
[0083] Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Chronic inflammation is widely observed in obesity. Understanding the molecular basis of inflammation has led to the identification of markers that may also serve as new targets of therapy in the management of associated cardiometabolic syndrome disease in obese person. The obese commonly have many elevated markers of inflammation, including: Interlukins (IL 6, 8 and 18), TNF- (Tumor necrosis factor-alpha), CRP (C-reactive protein), Insulin, Blood glucose, and Leptin. Inflammatory markers have been shown to predict future cardiovascular events in subjects with and without established cardiovascular disease (CVD).
[0084] Low-grade chronic inflammation is characterized by a two- to threefold increase in the systemic concentrations of cytokines such as TNF-, IL-6, and/or CRP. TNF's primary role is to regulate the immune cells and induce inflammation. TNF-induced reductions in insulin sensitivity in adipocytes are partly responsible for the increased free fatty acid production and hypertriglyceridaemia characteristic of abdominal obesity. Leptin responds specifically to adipose-derived inflammatory cytokines. Hyperglycemia induces IL-6 production from endothelial cells and macrophages. Meals high in saturated fat, as well as meals high in calories have been associated with increases in inflammatory markers.
[0085] Liver plays an important role in metabolism activities and it is an important site of fat metabolism. When this function is impaired due to a variety of reasons, fat accumulation occurs in the liver, which may result in cirrhosis and/or increased risk of other cardiometabolic syndromes such as for example diabetes, hypertension, disturbed lipid profile, and/or one or more risk factors associated with these syndromes, or in combination with other associated conditions.
[0086] As per one embodiment, the methods described herein are comprised of administering beta-cryptoxanthin compositions to a subject in need thereof, in an effective amount, and evaluating its effect on risk factors associated with cardiometabolic syndrome. Beta-cryptoxanthin compositions herein may be administered by oral route, in combination with antioxidant or other nutrients, using oil vehicle for suspending the composition. The oil used in the composition is selected from the group consisting of rape seed oil, corn oil, sunflower oil and like thereof.
[0087] According to one embodiment, methods and compositions as described herein are directed to treating macular degeneration in a subject in need thereof comprising essentially of administering therapeutically active amounts of beta-cryptoxanthin either alone or in combination with antioxidant or an oil.
[0088] As per one embodiment, the compositions and methods herein can improve (e.g. reduce) risk factors associated with cardiometabolic syndrome, such as body weight, lipid profile, body glucose, and/or insulin resistance, when administered to a subject, such as for example a subject who is fed with a high fat diet.
[0089] In another embodiment, a method for treating dyslipidema, comprising identifying a subject with elevated triglycerides levels, elevated serum LDL levels, or reduced HDL levels and accordingly administering a therapeutically effective amount of a composition consisting essentially of beta-cryptoxanthin either alone or in combination of pharmaceutically acceptable excipients.
[0090] In further embodiment, methods described herein are comprised of administering effective amount of a composition to a subject in need thereof for improving insulin sensitivity. The composition may be beta-cryptoxanthin either alone or in combination of pharmaceutically acceptable excipients.
[0091] The terminology high fat diet as used in the specification includes a diet with food typically containing about 32 to 60% of calories from fat. Such diets with 60 kcal % fat are commonly used to induce obesity in rodents since animals tend to gain weight more quickly, thereby allowing researchers to screen their compounds after a shorter period of time.
[0092] The type of fat is also considered when choosing a high-fat diet for an animal study. Many high-fat diets used in laboratory animal research contain more saturated fat such as lard, beef tallow, or coconut oil and these diets are quite capable of inducing obesity in susceptible strains.
[0093] As per one embodiment, methods described herein are comprised of administering an effective amount of beta-cryptoxanthin compositions to treat hyperlipidemia in a subject in need thereof by lowering total cholesterol, low density lipoproteins and/or triglycerides.
[0094] According to one embodiment, methods and compositions described herein are directed to lowering free fatty acid levels, and/or visceral fat, along with liver weight and body weight, when administered to a subject, who may be fed with a high fat diet.
[0095] According to one embodiment, methods and beta-cryptoxanthin compositions herein are also used to treat and/or evaluate their effect on expression of inflammatory markers and/or oxidative stress markers. It is observed that beta-cryptoxanthin compositions herein and methods of use thereof reduce inflammatory markers.
[0096] According to one embodiment, beta-cryptoxanthin compositions and methods of use thereof can protect organs, which may be at risk because of cardiometabolic syndrome, such as the eye and liver by reducing oxidative stress and/or inflammatory manifestations.
[0097] In one embodiment, beta-cryptoxanthin compositions and methods herein are directed to treat and/or be evaluate for their effect on the management of risk factors associated with cardiometabolic syndrome, in a subject, in need thereof, when administered in an effective amount(s).
[0098] In another embodiment, as per methods described herein, beta-cryptoxanthin compositions described herein are evaluated for effectiveness in significantly overcoming the cardiometabolic syndrome and associated risk factors such as body weight, body fats, lipid profile, blood glucose and the like.
[0099] The methods as described herein are comprised of evaluating effect of beta-cryptoxanthin composition on prevention and treatment of cardiometabolic syndrome through gene expression study on adipocyte cell system. CCAAT/enhancer binding proteins (C/EBP) are involved in different cellular responses, such as in the control of cellular proliferation, growth and differentiation, in metabolism, and in immunity. Their expression is regulated at multiple levels, including hormones, mitogens, cytokines, nutrients, and other factors. The encoded protein has been shown to bind to the promoter and modulate the expression of the gene encoding leptin, a protein that plays an important role in body weight homeostasis. C/EBP is involved in adipogenesis and with normal adipocyte function. C/EBP promotes adipogenesis by inducing the expression of PPAR.
[0100] Fatty Acid Synthase main function is to catalyze the synthesis of palmitate from acetyl-CoA and malonyl-CoA, in the presence of nicotinamide adenine dinucleotide (NADPH), into long-chain saturated fatty acids. The role of fatty acid synthase is implicated in the regulation of fatty acid synthesis and net accumulation of lipid in liver and adipose tissue. The role of fatty acid synthase is implicated in the regulation of fatty acid synthesis and net accumulation of lipid in liver and adipose tissue. FAS expression was controlled possibly at transcriptional level through peroxisome proliferator-activated receptor (PPARs) and sterol regulatory element-binding proteins (SREBPs) mediated signaling path way.
[0101] Stearoyl-CoA desaturase-1 (SDC-1) is a key enzyme in fatty acid metabolism. The elevated expression levels of SCD1 are found to be correlated with obesity. This phenomenon depends on increased expression of fatty acid biosynthetic enzymes that produce required fatty acids in large quantities. Alteration in SCD1 expression changes the fatty acid profile of these lipids and produces diverse effects on cellular function. High SCD1 expression is correlated with metabolic diseases such as obesity and insulin resistance, whereas low levels are protective against these metabolic disturbances. However, SCD1 is also involved in the regulation of inflammation and stress in distinct cell types, including -cells, adipocytes, macrophages, endothelial cells, and myocytes.
[0102] Particularly the effect of the beta-cryptoxanthin compositions are evaluated for fat accumulation, modulation of collagen and transforming growth factor beta (TGF-beta) signaling pathways in high fat fed diet (HFFD) rats. The effect of beta-cryptoxanthin compositions are also evaluated on vascular endothelial growth factor (VEGF), nuclear factor erythroid 2 (NrF2), nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), Inducible nitric oxide synthase (INOS), intercellular adhesion molecule 1 (ICAM-1) and heme oxygenase 1 (HO-1) pathways in retinal tissue of HFD treated rats.
[0103] Additionally the beta-cryptoxanthin compositions are evaluated on beta-carotene oxygenase 2 (BCO2), tumor necrosis factor alpha (TNF-), peroxisome proliferator-activated receptor gamma (PPAR-), nuclear factor erythroid 2 (Nrf-2), nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), insulin receptor substrate 1 (IRS-1), heme oxygenase 1 (HO-1) pathways in liver tissue of HFD treated rats.
[0104] The compositions herein include beta-cryptoxanthin concentrates of high purity. In particular, beta-cryptoxanthin concentrates containing about 10-80% by weight total xanthophylls (total carotenoids) of which the trans-beta-cryptoxanthin content is about 75-98% by weight and the remaining including zeaxanthin, trans-capsanthin, beta-carotene and trace amounts of other carotenoids. The concentrates are particularly useful as dietary supplements for nutrition and health promoting benefits.
[0105] Processes are described for the preparation of the beta-cryptoxanthin concentrate from plant oleoresin, especially from Capsicum oleoresin. The process includes the steps of admixing the oleoresin with alcohol solvents, saponifying the xanthophyll esters, washing and purifying by eluting the crude xanthophyll viscous concentrate on a silica gel column, and purifying further by washings to obtain high purity trans-beta-cryptoxanthin enriched concentrate crystals.
[0106] In some embodiments, a process is described for the isolation of beta-cryptoxanthin crystals containing at least or about 80% by weight of total xanthophylls (total carotenoids) in free form, out of which the trans-beta-cryptoxanthin content is at or about or at least 98.5% by weight, the remaining including trace amounts of zeaxanthin, trans-capsanthin, beta-carotene and other carotenoids derived from oleoresin and extracts of plant materials such as Capsicum sources.
[0107] In some embodiments, a process is described for the preparation of beta-cryptoxanthin crystals containing at or about or at least 40% by weight of total carotenoids, out which the trans-beta-cryptoxanthin is at or about or at least 90% by weight, the remaining including trace amounts of zeaxanthin, trans-capsanthin, beta-carotene and other carotenoids derived from oleoresin and extracts of plant materials such as Capsicum sources.
[0108] In some embodiments, a process is described for the preparation of beta-cryptoxanthin crystals containing at or about or at least 10% by weight of total carotenoids, out of which the trans-beta-cryptoxanthin is at or about or at least 75% by weight, the remaining including zeaxanthin, trans-capsanthin, beta-carotene and traces amounts of other carotenoids derived from oleoresin and extracts of plant materials such as Capsicum sources.
[0109] In some embodiments, a process is described for the preparation of beta-cryptoxanthin crystals containing total carotenoids at or about 10 to at or about 80% by weight, out of which the trans-beta-cryptoxanthin content is in the range of at or about 75 to at or about 98% by weight, the rest including zeaxanthin, trans-capsanthin, beta-carotene and trace amounts of other carotenoids derived from a starting material like saponified Capsicum extract.
[0110] In some embodiments, a process is described for the preparation of high purity beta-cryptoxanthin from capsicum oleoresin or saponified capsicum extract. In some embodiments, residual solvent-free beta-cryptoxanthin crystals, in which trans-beta-cryptoxanthin form the major ingredient in the total carotenoids. In one embodiment, processes herein provide recovery of carotene hydrocarbon fractions rich in beta-carotene.
[0111] In some embodiments, the process for obtaining high purity trans-beta-cryptoxanthin includes: [0112] saponification of esterified xanthophylls in Capsicum extract, which results in free xanthophylls and which is purified by washing with acidified water, followed by drying to obtain a carotenoid mass; [0113] treating the carotenoid mass with non-polar solvent under stirring, followed by filtration and concentration to obtain a mass; [0114] subjecting the mass to column chromatography using silica gel and elution using non-polar solvent to remove beta-carotene; [0115] eluting the column with non-polar solvent containing at or about 2% polar solvent, and obtaining an eluent after concentration of a concentrate showing at or about 10% total carotenoids by weight, of which trans -beta-cryptoxanthin comprises at or about 75% by weight; [0116] treating the above concentrate with ethanol under stirring, followed by cooling to at or about 10 C. and filtering to obtain a semi -purified crystalline mass showing total xanthophylls at or about 40% by weight, of which trans-beta-cryptoxanthin comprises at or about 98%> by weight; and [0117] washing the crystalline mass with hexane containing about 20% ethyl acetate, cooling to at or about 10 C. and filtering to obtain a high purity crystalline material showing at or about 80% total xanthophylls by weight, of which trans-beta-cryptoxanthin comprises at or about 98.5%> by weight.
[0118] In some embodiments, a process is described for the preparation of a beta-cryptoxanthin enriched concentrate from plant material comprising at or about 10-80% by weight total xanthophylls, of which at or about 75-98% by weight is trans-beta-cryptoxanthin. The process comprises: (a) mixing an oleoresin of plant material comprising xanthophylls esters with an aliphatic alcoholic solvent; (b) saponifying the xanthophylls esters present in the oleoresin with an alkali at an elevated temperature; (c) removing the aliphatic alcoholic solvent followed by addition of water to obtain a diluted resultant mixture; (d) adding a diluted organic acid to the diluted resultant mixture to form a water layer and a precipitated xanthophylls mass; (e) removing the water layer and washing the precipitated xanthophylls mass with a polar solvent; (f) drying the precipitated xanthophylls mass to obtain a crude xanthophylls mass; (g) washing the crude xanthophylls mass with a non-polar solvent and concentrating the non-polar solvent washings to obtain a concentrated crude xanthophylls mass; (h) transferring the concentrated crude xanthophylls mass to a silica gel column and washing with a non-polar solvent; (i) eluting the column with a mixture of non-polar and polar solvent and concentrating the elutions to obtain a trans-beta-cryptoxanthin-rich xanthophylls concentrate; (j) admixing the trans-beta-cryptoxanthin-rich xanthophylls concentrate with an aliphatic alcohol and then cooling; and (k) filtering and drying the trans-beta-cryptoxanthin-rich xanthophylls concentrate to obtain a purified trans-beta-cryptoxanthin concentrate.
[0119] In some embodiments, the xanthophylls esters in the oleoresin of plant material in step (a) are present at or about 6-8% by weight. In some embodiments, the aliphatic alcohol of step (a) or (j) is selected from the group consisting of ethanol, methanol, isopropyl alcohol, and mixtures thereof.
[0120] In some embodiments, the ratio of oleoresin to alcohol in step (a) ranges from at or about 1:0.25 to at or about 1:1 weight/volume. In some embodiments, the alkali of step (b) is selected from the group consisting of sodium hydroxide, potassium hydroxide, and mixtures thereof. In some embodiments, the ratio of oleoresin to alkali in step (b) ranges from at or about 1:0.25 to at or about 1:0.5 weight/weight.
[0121] In some embodiments, the elevated temperature of step (b) ranges from at or about 75 to at or about 85 C. In some embodiments, the addition of water in step (c) is at or about 5 times that of the oleoresin (weight/weight).
[0122] In some embodiments, the diluted organic acid of step (d) is acetic acid or phosphoric acid. In some embodiments, the diluted organic acid of step (d) is a solution of at or about 20% to at or about 50% organic acid.
[0123] In some embodiments, the polar solvent of step (e) is water.
[0124] In some embodiments, the non-polar solvent of steps (g), (h), and (i) is selected from the group consisting of a hexane, a pentane, a heptane, and mixtures thereof.
[0125] In some embodiments, the crude xanthophylls mass and non-polar solvent of step (g) is in a ratio of at or about 1:10 to at or about 1:15 weight/volume. In some embodiments, the concentrated crude xanthophylls mass of step (g) comprises beta-carotene, trans-beta-cryptoxanthin, trans-capsanthin, zeaxanthin, and trace amounts of other carotenoids, such as capsorubin or violaxanthin.
[0126] In some embodiments, the concentrated crude xanthophylls mass and the non-polar solvent of step (h) are in a ratio of at or about 1:5 to at or about 1:8 weight/volume. In some embodiments, a carotene concentrate is obtained by distilling the non-polar solvent washing of step (h). In some embodiments, the carotene concentrate is beta-carotene.
[0127] In some embodiments, the polar solvent of step (i) is selected from the group consisting of a propanone, a pentanone, and mixtures thereof. In some embodiments, the non-polar solvent and polar solvent of step (i) are in a ratio of at or about 95:5 to at or about 98:2. In some embodiments, the trans-beta-cryptoxanthin-rich xanthophylls concentrate of step (i) comprises at or about or at least 10% by weight of total xanthophylls, of which trans-beta-cryptoxanthin content is at or about or at least 75% by weight.
[0128] In some embodiments, the cooling in step j) is performed at or about 10 C. In some embodiments, the purified trans-beta-cryptoxanthin concentrate of step (k) comprises at or about or at least 40% by weight of total xanthophylls, of which trans-beta-cryptoxanthin content is at or about or at least 90% by weight.
[0129] In some embodiments, the process further comprises a step (1): washing the purified trans-beta-cryptoxanthin concentrate with a mixture of non-polar and ester solvent and cooling for precipitation to obtain high purity trans-beta-cryptoxanthin crystals. In some embodiments, the high purity trans-beta-cryptoxanthin crystals of step (1) comprises at or about or at least 80% by weight of total xanthophylls, of which trans-beta-cryptoxanthin content is at or about or at least 98% by weight. In some embodiments, the ester solvent of step (1) is ethyl acetate and the non-polar solvent of step (1) is hexane. In some embodiments, the non-polar solvent and ester solvent of step (1) are in a ratio of at or about 80:20 to at or about 90:10. In some embodiments, the temperature for cooling in step (1) is at or about 10 C.
[0130] In some embodiments, a process is described for the preparation of a beta-cryptoxanthin enriched concentrate from plant material comprising at or about or at least 80% by weight total xanthophylls, of which at or about or at least 98% by weight is trans-beta-cryptoxanthin, the process comprising: (a) mixing an oleoresin of plant material comprising xanthophylls esters with ethanol, wherein the ratio of oleoresin to ethanol is at or about 1:1 weight/volume; (b) saponifying the xanthophylls esters present in the oleoresin with potassium hydroxide without addition of water, wherein the ratio of oleoresin to potassium hydroxide is at or about 1:0.25 weight/weight; (c) applying heat to the oleoresin to elevate the temperature up to reflux at or about 80-85 C.; (d) agitating the oleoresin for about 3 to 5 hours at or about 80-85 C.; (e) evaporating the ethanol under vacuum followed by addition of water at or about 5 times that of the oleoresin (weight/weight) to obtain a diluted resultant mixture and agitating for at or about 1 hour; (f) neutralizing the diluted resultant mixture with about 25% acetic acid to form a water layer and a precipitated xanthophylls mass; (g) separating the water layer from the precipitated xanthophylls mass and washing the mass with water to remove soaps and other polar soluble materials; (h) drying the precipitated xanthophylls mass under vacuum to obtain a crude xanthophylls mass; (i) washing the crude xanthophylls mass with at or about 1:10 hexane (weight/volume) and concentrating the hexane washings to obtain a concentrated crude xanthophylls mass; j) transferring the concentrated crude xanthophylls mass to a silica gel column at a ratio of at or about 1:5 (weight/weight) and eluting with hexane to obtain a carotene fraction; (k) washing the column with at or about 98:2 hexane to acetone and concentrating the washings to obtain a trans-beta-cryptoxanthin-rich xanthophylls concentrate; (l) admixing the trans-beta-cryptoxanthin-rich xanthophylls concentrate with at or about 1:2 ethanol under stirring and then cooling at or about 10 C. for about 8 hours; (m) filtering and drying the trans-beta-cryptoxanthin-rich xanthophylls concentrate under vacuum to obtain a purified trans-beta-cryptoxanthin concentrate; and (n) washing the purified trans-beta-cryptoxanthin concentrate with at or about 80:20 hexane :ethyl acetate and cooling to at or about 10 C. for about 18 hours for precipitation to obtain high purity trans-beta-cryptoxanthin crystals.
[0131] In some embodiments, the total xanthophylls of the processes comprise byproducts selected from zeaxanthin, trans-capsanthin, beta-carotene, trace amounts of other carotenoids, and any combinations thereof.
[0132] In some embodiments, the plant material used in the processes or to derive the beta-cyrptoxanthin concentrates is selected from at least one of the group consisting of fruits, vegetables, and mixtures thereof. In some embodiments, the plant material is from a capsicum.
[0133] In some embodiments, beta-cryptoxanthin concentrates herein may be administered in a dosage form selected from beadlets, microencapsulated powders, oil suspensions, liquid dispersions, capsules, pellets, ointments, soft gel capsules, tablets, chewable tablets or lotions/liquid preparations. In some embodiments, the beta-cryptoxanthin concentrate is added to or as part of another composition.
[0134] In some embodiments, compositions herein includes a beta-cryptoxanthin concentrate derived from plant material, wherein the concentrate comprises at or about or at least 10% by weight total xanthophylls, of which at or about or at least 75% by weight is trans-beta-cryptoxanthin. In some embodiments, the total xanthophylls comprise by-products selected from zeaxanthin, trans-capsanthin, beta-carotene, trace amounts of other carotenoids such as capsorubin or violaxanthin, and combinations thereof. In some embodiments, the composition further comprises a pharmaceutically acceptable ingredient or a food grade ingredient.
[0135] In some embodiments, the total xanthophylls of the beta-cryptoxanthin concentrate comprise by-products selected from the group consisting of zeaxanthin, trans-capsanthin, beta-carotene, trace amounts of other carotenoids such as capsorubin or violaxanthin, and combinations thereof.
[0136] In some embodiments, a beta-cryptoxanthin concentrate is provided, which contains at or about 10-80% by weight total xanthophylls, of which at or about 75-98% by weight is trans-beta-cryptoxanthin, the remaining including zeaxanthin, trans-capsanthin, beta-carotene and trace amounts of other carotenoids, derived from oleoresin or extract of plant material and which is useful for nutrition and health care. In some embodiments, the concentrate comprises at or about or at least 10% by weight total xanthophylls, of which at or about or at least 75% by weight is trans-beta-cryptoxanthin. In some embodiments, the concentrate comprises at or about or at least 40% by weight total xanthophylls, of which at or about or at least 90% by weight is trans-beta-cryptoxanthin.
[0137] In some embodiments, the concentrate comprises at or about or at least 80% by weight total xanthophylls, of which at or about or at least 98% by weight is trans-beta-cryptoxanthin.
[0138] The plant material is derived from sources including, but not limited to, fruits and vegetables. In some embodiments, the plant material is derived from capsicum. Capsicum is a genus of flowering plants that includes several varieties of peppers, such as but not limited to red peppers, and the word capsicum is also used interchangeably in several parts of the world when referring to peppers. The capsicum oleoresin described herein also includes paprika oleoresin.
[0139] In some embodiments, beta-cryptoxanthin enriched concentrates herein can be formulated in a dosage form including, but not limited to, beadlets, microencapsulated powders, oil suspensions, liquid dispersions, capsules, pellets, ointments, soft gel capsules, tablets, chewable tablets or lotions/liquid preparations. The beta-cryptoxanthin enriched concentrates herein can also be provided in a food or feed (including liquid or solid) composition. It will be appreciated that suitable delivery methods include, but are not limited to, oral, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, intracranial, or buccal administration.
[0140] Compositions herein comprising the trans-beta-cryptoxanthin enriched concentrates herein include in some embodiments one or more suitable pharmaceutically acceptable ingredients or food grade ingredients such as, but not limited to, carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrators, solubilizers and isotonic agents.
[0141] Compositions herein may include an effective amount of the trans-beta-cryptoxanthin enriched concentrates. An effective amount refers to an amount effective, at a dose and in certain circumstances for a period of time to achieve a desired result, for example in methods of treatment or prevention of symptoms for use in such methods. The effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual.
[0142] The beta-cryptoxanthin compositions herein includes an active material present including beta-cryptoxanthin (BCX), which is extracted for example from paprika oleoresin by saponification followed by purification through column chromatography. Compositions herein are enriched with trans-beta-cryptoxanthin. In an embodiment, the extract is suspended in a suitable oil medium to obtain 5% oil suspension. In an embodiment, the suspension was evaluated in animal model described herein below. For human consumption, the compositions herein include final formulations into powders, granules, beadlets, and can be administered by oral solid dosage forms such as tablets, capsules.
[0143] In an embodiment, an effective amount herein relates to the amount of BCX present in the composition.
[0144] In an embodiment, a daily dose duration can range from at or about 3 months to at or about 2 years, or till the desired effect is achieved in a subject. It will be appreciated that there may be no fixed time period for the daily doses as it may be less or longer than such range. It will also be appreciated that the dose may be given continuously daily during this period or the administration can be stopped after obtaining a desired effect in a subject, and can also be restarted again as needed. It is appreciated that dose periods herein include the experiment durations or by general volunteer study period which can be extended to 12 months.
[0145] In an embodiment, an effective daily dose includes a range of at or about 250 micrograms to at or about 30 mg/kg body weight.
[0146] In an embodiment, an effective daily dose includes a range of at or about 150 micrograms to at or about 20 mg/kg body weight.
[0147] In an embodiment, an effective daily dose includes a range of at or about 200 micrograms to at or about 10 mg/kg body weight.
[0148] Compositions and methods of preparing beta-cryptoxanthin are disclosed in Applicant's copending published application US 2015/0361040, which is herewith incorporated by reference.
[0149] The following examples are given by the way of illustration and therefore should not be construed to limit the scope of the disclosures or innovations herein.
[0150] While the compositions and methods have been described in terms of illustrative embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the compositions and methods herein. The details and advantages of which are explained hereunder in greater detail in relation to non-limiting exemplary illustrations.
EXAMPLES
[0151] Process for Extraction of Beta-Cryptoxanthin from Paprika Oleoresin
Example 1
[0152] A weighed quantity of 100 g of Paprika oleoresin containing 7.72% total xanthophylls and a color value of 1,23,515 units (HPLC profile of the oleoresin: beta-15.36% carotene; 10% trans-beta-cryptoxanthin; 7.6% zeaxanthin; and 31.50% trans-capsanthin) was mixed with 100 ml ethanol and 25 g potassium hydroxide pellet. The reaction mixture was heated to a temperature of 80-85 C. with stirring. This saponification process was maintained for 3-5 hours at 80-85 C. with gentle agitation. The reaction mixture was cooled, and then ethanol was distilled out from the mass. A measured volume of water (700 ml) was added to the reaction mixture and agitated for 1 hour. The solution was neutralized with 25% acetic acid solution. The water layer from the mass was separated, and the mass was washed thrice with water. The mass was collected and dried under vacuum. The saponifed mass concentrate obtained was 124 g with a total xanthophylls content of 3.73% by weight (HPLC profile of the saponifed mass concentrate: 22.53% beta-carotene; 12.32% trans-beta-cryptoxanthin; 11% zeaxanthin; and 29.3% trans-capsanthin).
[0153] The saponified mass concentrate was washed two times with 1:10 hexane (wt/vol) at room temperature under stirring, filtered, and the combined filtrate concentrated to obtain a concentrated crude xanthophylls mass. The concentrated crude xanthophylls mass (hexane concentrate) obtained was 72 g with a total xanthophylls content of 3.2% (HPLC profile of the concentrated crude xanthophylls mass: 39.01% beta-carotene; 21.78% trans-beta-cryptoxanthin; 5.70% zeaxanthin; and 9.86% trans-capsanthin).
[0154] The residue (saponified xanthophylls) remaining after hexane wash was 22 g, which on analysis showed a total xanthophylls content of 10% (HPLC profile of the residue: 0.7% beta-carotene; 3.43% trans-beta-cryptoxanthin; 15.32% zeaxanthin; and 52.84% trans-capsanthin).
[0155] The hexane concentrate was dissolved in a minimum amount of hexane and subjected to column chromatographic separation. The column was packed with 1:5 concentrate to Silica 100-200 mesh (wt/wt). The column was washed with hexane, and the separated band was collected and concentrated (yield 55 g with a total xanthophylls content of 2.3%, HPLC profile: 99.8% beta-carotene). The column was then eluted with 98:2 hexane: acetone (v/v), and the eluent collected and concentrated. This concentrate layer was enriched with beta-cryptoxanthin (yield 5.2 g with a total xanthophylls content of 10.26%, HPLC profile: 75.56% trans-beta-cryptoxanthin). Finally, the column was washed with acetone and the washings concentrated to obtain trans-capsanthin enriched residue.
Example 2
[0156] A quantity of approximately 100 g of Paprika oleoresin containing 6.50% total xanthophylls and a color value of 1,05,457 units (HPLC profile of the oleoresin: 15.73% beta-carotene; 9.07% trans-beta-cryptoxanthin; 10.54% zeaxanthin and 31.38% trans-capsanthin) was mixed with 100 ml ethanol and 25 g potassium hydroxide pellet. The reaction mixture was heated to a temperature of 80-85 C. with stirring. This saponification process was maintained for 3-5 hours at 80-85 C. with gentle agitation. The reaction mixture was cooled, and then ethanol was distilled out from the mass. A measured volume of water (700 ml) was added to the reaction mixture and agitated for 1 hour. The solution was neutralized with 40% acetic acid solution. The water layer from the mass was separated, and the mass was washed thrice with water. The mass was collected and dried under vacuum. The saponified mass concentrate obtained was 126 g with a total xanthophylls content of 3.73% by weight (HPLC profile of the saponified mass concentrate: 16.34% beta-carotene; 9.41% trans-beta-cryptoxanthin; 8.57% zeaxanthin; and 24.35% trans-capsanthin).
[0157] The saponified mass concentrate was washed two times with 1:10 hexane (wt/vol) at room temperature under stirring, filtered, and the combined filtrate concentrated to obtain a concentrated crude xanthophylls mass. The concentrated crude xanthophylls mass (hexane concentrate) obtained was 76.15 g with a total xanthophylls content of 3.26% (HPLC profile of the concentrated crude xanthophylls mass: 31.80% beta-carotene; 14.04% trans-beta-cryptoxanthin; 4.35% zeaxanthin; and 8.70% trans-capsanthin).
[0158] The residue (saponified xanthophylls) remaining after hexane wash was 16 g, which on analysis showed a total xanthophylls content of 11% (HPLC analysis of the residue: 1.22% beta-carotene; 0.75% trans-beta-cryptoxanthin; 33.29% zeaxanthin; and 29.99% trans-capsanthin).
[0159] The hexane concentrate was dissolved in a minimum amount of hexane and subjected to column chromatographic separation. The column was packed with 1:5 concentrate to Silica 100-200 mesh (wt/wt), eluted with hexane, and the first band separated was collected and concentrated (yield 54.72 g with a total xanthophylls content of 1.08%, HPLC profile: 85.88% beta-carotene). The column was then eluted with 98:2 hexane: acetone (v/v) collecting the eluent fraction and concentrated. This fraction was enriched with beta-cryptoxanthin, yielding 4.02 g with a total xanthophylls content of 9% (HPLC profile of the enriched beta-cryptoxanthin concentrate: 76.04% trans-beta-cryptoxanthin). Finally the column was washed with acetone.
[0160] The 4.02 g fraction concentrate was stirred with 1:2 ethanol (wt/vol) for 1 hr, chilled for 8 hrs at 10 C., filtered, and the precipitate dried under vacuum. The yield obtained was 0.42 g crystalline precipitate with a total xanthophylls content of 42.45%. The HPLC profile of the crystalline precipitate showed 98.3% trans-beta-cryptoxanthin.
Example 3
[0161] A weighed quantity of Paprika oleoresin (100 g) containing 6-8%> by weight total xanthophylls and a color value of 100,000 units (HPLC profile of the oleoresin: 15.36% beta-carotene; 10% trans-beta-cryptoxanthin; 7.6% zeaxanthin; and 31.50% trans-capsanthin) was mixed with 100 ml ethanol and 25 g potassium hydroxide pellet. The reaction mixture was heated to a temperature of 80-85 C. with stirring. This saponification process was maintained for 3-5 hours at 80-85 C. with gentle agitation. The reaction mixture was cooled and then ethanol was distilled off from the mass under vacuum. A measured volume of water (700 ml) was added to the reaction mixture and agitated for 1 hour. The solution was neutralized with 25% acetic acid solution. The water layer from the mass was removed, and the mass was washed thrice with water. The mass was collected and dried under vacuum. The saponified mass concentrate obtained was 121.75 g with a total xanthophylls content of 4.92% by wt (HPLC profile of the saponified mass concentrate: 21.76% beta-carotene; 12.74% trans-beta-cryptoxanthin; 10.13% zeaxanthin; and 38.25% trans-capsanthin).
[0162] The saponified mass concentrate was washed two times with 1:10 hexane (wt/vol) at room temperature under stirring, filtered, and the combined filtrate concentrated to get a concentrated crude xanthophylls mass. The concentrated crude xanthophylls mass (hexane concentrate) obtained was 85.81 g with a total xanthophylls content of 3.21% by wt (HPLC profile of the concentrated crude xanthophylls mass: 35.28% beta-carotene; 19.65% trans-beta-cryptoxanthin; 3.99% zeaxanthin; and 13.88% trans-capsanthin).
[0163] The residue (saponified xanthophylls) remaining after hexane wash was 25.65 g, which on analysis showed a total xanthophylls content of 10.42% by wt (HPLC analysis of the residue: 0.7% beta-carotene; 1.24% trans-beta-cryptoxanthin; 18.98% zeaxanthin; and 52.32% trans-capsanthin.
[0164] The hexane concentrate was dissolved in minimum amount of hexane and subjected to column chromatographic separation. The column was packed with 1 :5 concentrate to Silica gel 100-200 mesh (wt/wt), eluted with 5-8 volumes of hexane, and the first band separated was eluted and concentrated (yield 55 g with a total xanthophylls content of 2.29% wt, HPLC profile: 99% beta-carotene). The column was then eluted with 98:2 hexane: acetone (vol/vol) collecting the eluent fraction and concentrated. This concentrate was enriched with beta-cryptoxanthin, yielding 9.06 g with a total xanthophylls content of 6.12% by wt (HPLC profile of the enriched beta-cryptoxanthin concentrate: 71.80% trans-beta-cryptoxanthin). Finally the column was eluted with acetone.
[0165] The 9.06 g beta-cryptoxanthin concentrate was stirred with 1:2 ethanol (wt/vol) for 1 hr, chilled for 8 hours at 10 C., filtered, and the precipitate dried under vacuum. The yield obtained was 0.5 g with a total xanthophylls content of 42.35% by wt. The HPLC profile of the crystal showed 98.3% trans-beta-cryptoxanthin content.
[0166] The 0.5 g beta-cryptoxanthin precipitate was dissolved in a minimum amount of 80:20 hexane:ethyl acetate (vol/vol) and chilled for 18 hrs at 10 C., filtered, and the precipitate dried under vacuum. The yield obtained was 0.03 g with a total xanthophylls content of 80% and HPLC profile for trans-beta-cryptoxanthin of 98.50%.
Example 4
[0167] In-Vitro Evaluation of Beta-Cryptoxanthin Composition
[0168] 3T3L1 murine adipocytes model system was used to understand basic cellular mechanisms associated with diabetes, obesity and related disorders. Nutrigenomics study was carried out to evaluate effect of beta-cryptoxanthin on adipocyte cells, particularly differentiated 3T3-L1 cells, the study was based on real-time polymerase chain reaction (Real time PCR) with dose 12.5 ug/mL. The effect of beta-cryptoxanthin on Adipocyte differentiation and its activity for bio markers PPARG, SCD-1, acetyl Coa carboxylase (ACC), SREBP-1, C/EBP and FAS were evaluated.
[0169] It was observed that BCX down regulated C/EBP and fatty acid synthase (FAS) . BCX also down regulated Stearoyl-CoA desaturase-1 (SCD-1), wherein down-regulation of SCD-1 is an important component of leptin's metabolic actions. See
Example 5
[0170] In-Vivo Evaluation of Beta-Cryptoxanthin Composition in Rat
[0171] a. Effect of Beta-Cryptoxanthin Compositions on Cardiometabolic Markers, Fat Accumulation, Modulation of Collagen and TGF-Beta Signaling Pathways in High Fat Fed Diet (HFFD) Rats
[0172] Animals
[0173] Male Sprague Dawley rats (7 rats/group, age: 8 week, weight: 18020 g) were housed in a controlled environment with a 12:12-h light-dark cycle at 22 C. and provided with rat chow and water ad libitum. All experiments were conducted under the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals and approved by the Ethics Committee of the Veterinary Control Institute. After acclimation for 2 weeks, the rats were divided into four groups: Rats were randomly divided into the following groups: (1) Control, (2) High Fat Diet (HFD) (42% of calories from fat), (3) control+beta-cryptoxanthin (2.5 mg/kg) 4) HFD (42% of calories from fat)+beta-cryptoxanthin (2.5 mg/kg) was administered daily as supplement for 12 weeks.
[0174] i) Effect of BCX on Visceral Fat:
[0175] Visceral fat decreased in HFD treated BCX rats only. HFD increased visceral fat. Similarly decrease in body weight was observed in HFD treated BCX rats. Liver weight decreased in HFD treated BCX rats. (Table 1,
TABLE-US-00001 TABLE 1 Control BC HFD BC Initial BW (g) 233.86 232.71 232.29 234.29 Final BW (g) 286.29 290.29 340.86 325.43 Feed intake (g/d) 23.54 23.88 19.16 16.87 Visceral fat (g) 7.79 7.75 29.54 14.93 Liver (g) 12.29 12.74 20.56 18.46
[0176] Consumption of HF diet produced a significant (P<0.001) increase in body weight (BW) compared to the consumption of normal diet (normal control group). Beta-cryptoxanthin supplementation significantly reduced the body weight as compared to the HFD control group (P<0.05). Visceral fat and liver weights were significantly higher in the HFD group as compared to the control group (P<0.05). In rats fed a HF diet supplemented with Beta-cryptoxanthin, a tendency towards a decrease in visceral fat was observed (P<0.05) (see
[0177] ii) Effect of BCX on Lipid and Lipoproteins:
[0178] BCX reflects its significant role in cholesterol lowering effects. According to Table 2 (
TABLE-US-00002 TABLE 2 ND HFD Details Control Control + BCX HFD HFD + BCX T-C (mg/ml) 50.80 48.64 83.14 60.86 HDL-C (mg/dl) 17.14 17.00 20.43 19.71 LDL-C (mg/dl) 26.71 25.00 46.43 36.43 TG (mg/dl) 27.29 20.57 50.43 36.70
[0179] iii) Effect of BCX on Metabolic Markers/Hormones:
[0180] According to the study glucose, insulin and FFA (Free Fatty Acid) level decreased in HFD treated BCX rats. On the other hand leptin was decreased significantly in HFD treated BCX and adiponectin was increased in HFD treated BCX. It was observed BCX inhibited the glucose-mediated changes in metabolic markers and lipid profile (see Table 3,
TABLE-US-00003 TABLE 3 Serum glucose, insulin, leptin, adiponectin and lipid profile in control and HFD rats Normal diet (ND) High Fat Diet (HFD) Item Control Control + BCX HFD HFD + BCX Glucose (mg/dl) 79.00 78.29 195.14 168.14 Insulin (ng/mL) 1.78 1.73 7.76 5.31 FFA (mM) 1.23 1.12 3.81 2.61 Leptin (ng/mL) 30.71 30.43 107.71 82.00 Adiponectin (mg/mL) 10.37 10.81 5.97 7.79
[0181] There was a significant (P<0.001) elevation in serum glucose, insulin, and leptin levels in HFD-induced obese rats compared with control rats.
[0182] Total cholesterol (T-C), free fatty acids (FFAs), triglycerides (TGs), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) were checked in serum of control and HFD-induced obese rats, respectively. The concentrations of serum lipid profiles were significantly increased in experimental obese rats as compared to the normal rats. Treatment with beta-cryptoxanthin significantly reduced the concentrations of serum glucose, insulin, leptin, lipids concentrations in obese rats but decreased adiponectin concentration in HFD rat (P<0.05) (see
[0183] iv) Effect of BCX on Oxidative Stress Markers:
[0184] Oxidative stress is significantly reduced in BCX treated rats in serum and liver. HFD rats had high thiobarbituric acid reactive substance (TBARS) and BCX treated HFD rats significantly reduced oxidative stress by reducing TBARS in retina and serum. BCX is known as provitamin A. These results further support its antioxidant activity in reducing oxidative stress markers and protects eyes and other associated conditions.
TABLE-US-00004 TABLE 4 ND HFD Item Control BC HFD BC Serum TBARS (nmol/mL) 0.87 0.67 1.88 1.64 Retinal TBARS (mol/mg protein) 80.37 62.67 228.14 186.43
[0185] b. Effect of Beta-Cryptoxanthin Compositions on Retinal Tissue in HFD Treated Rats
[0186] Beta-cryptoxanthin has several functions that are important for human health, including roles in antioxidant defense and cell-to-cell communication. Beta-cryptoxanthin is a precursor of vitamin A, which is an essential nutrient needed for eyesight, growth, development and immune response. Increase in reactive oxygen species (ROS) is one of the major retinal metabolic abnormalities associated with the development of diabetic retinopathy. NF-E2-related factor 2 (Nrf2), a redox sensitive factor, provides cellular defenses against the cytotoxic ROS. In stress conditions, Nrf2 dissociates from its cytosolic inhibitor, Kelch-like ECH-associated protein 1 (Keap1), and moves to the nucleus to regulate the transcription of antioxidant genes including the catalytic subunit of glutamylcysteine ligase (GCLC), a rate-limiting reduced glutathione (GSH) biosynthesis enzyme.
[0187] Ocular neovascularization (NV) is a major cause of the blindness associated with ischemic retinal disorders, such as proliferative diabetic retinopathy (PDR), retinopathy of prematurity (ROP), and age-related macular degeneration (AMD). Studies showed that nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), plays an important role in eye diseases such as glaucoma, ROP and AMD.
[0188] Animals fed on HFD showed an increased upregulation of inflammatory and proangiogenic markers. This animal model may be useful to study mechanisms of diabetic retinopathy and therapeutic targets.
[0189] According to
[0190] HO-1 is a sensitive marker for assessing light-induced insult in the retina. Increased expression of HO-1 is thought to be a cellular defense against oxidative damage, and its expression may play an important role in protecting the retina against light damage (see
[0191] Leukocytes play a critical role in ocular diseases such as uveitis, diabetic retinopathy, and choroidal neovascularization. Intercellular adhesion molecule (ICAM)-1 is essential for the migration of leukocytes. Control of ICAM-1 expression may lead to therapies for these diseases. Down regulation of ICAM-1 expression to reduce retinal neovascular disease by inhibiting leukocyte infiltration (see
[0192] To treat/prevent eye diseases like AMD, the effect of BCX on inhibition or induction of iNOS can be checked in animal models. BCX decreased iNOS and may be potential for neovascualarization (see
[0193] It is also observed that BCX inhibited the glucose-mediated induction of NF-kB expression in retina (see
[0194] Nrf2 is involved in the cytoprotective mechanism in the retina in response to ischemia-reperfusion injury and suggests that pharmacologic induction of Nrf2 could be a new therapeutic strategy for retinal ischemia-reperfusion and other retinal diseases (see
[0195] VEGF has been considered to be a mediator of diabetic retinopathy. Inhibition of VEGF reduces retinal neovascularization (see
[0196] c. Effect of Beta-Cryptoxanthin Compositions on Liver Tissue in HFD Treated Rats
[0197] Animals and Diets
[0198] The experiment was performed using 28 male Sprague-Dawley rats (8 weeks old, weighing 18020 g), purchased from the Inonu University Laboratory Animal Research Center (Malatya, Turkey). Rats were housed in cages in a temperature and humidity controlled environment, on a 12-hr light and 12-hr dark cycle, designed for the purpose of the study. The temperature inside the rat cages was 212 C., relative humidity was 555% and consecutive light-dark cycles lasted 12 hours. The protocol of the study was approved by the Animal Experimentation Ethics Committee of Inonu University (Malatya, Turkey). All procedures involving rats were conducted in strict compliance with relevant laws, the Animal Welfare Act, Public Health Services Policy, and guidelines established by the Institutional Animal Care and Use Committee of the Institute. Prior the starting the experiment, animals were assigned to either a regular diet (control; 12% of calories from fat) or a high-fat diet (HFD, 42% of calories from fat). Control or HFD composed according to the American Institute of Nutrition AIN-93 (Reeves et al., 1993) recommendations of casein (20%), soybean oil (7%), wheat starch (53.2%), sucrose (10%), potato starch (5%), 1-cysteine (0.3%), vitamin mix AIN-93M (1%) and mineral mix AIN-93M (3.5%). The high-fat diets (42% calories from fat) were obtained from the basal AIN-93 diet, by replacement of wheat starch with fat (tallow 15% and soybean oil 10%). For induction of obesity (insulin resistance), the rats were fed with HFD for 12 weeks
[0199] Experimental Protocol
[0200] After a one week period of adaptation to laboratory conditions, the animals were randomly divided into four equal groups: 1) Control (n=7): untreated rats were allowed free access to a standard diet; 2) BCX Group, rats were fed a standard diet supplemented with beta-cryptoxanthin (n=7) 2.5 mg/kg; 3) HFD Group (high-fat diet; 42% of calories from fat; n=7): rats were fed a high-fat diet 4) HFD+BCX Group, rats were fed a high-fat diet (42% of calories from fat; n=7) supplemented with beta-cryptoxanthin 2.5 mg/kg. 5% suspension of beta-cryptoxanthin was dissolved in corn oil. At the end of the experiment, all rats were sacrificed by cervical dislocation. Blood samples were taken from rats in the morning upon overnight fasting for biochemical analyses and their visceral fat and liver samples were removed and weighed after sacrificing the animals.
[0201] Laboratory Measurements
[0202] Blood was collected by cardiac puncture using an anticoagulant-free vacutainer tube, later centrifuged at 3,000g for 10 min to obtain serum and kept frozen at 80 C. until it was assayed for biochemical parameters and malondialdehyde (MDA). Serum biochemical parameters were estimated using an automatic analyzer (Samsung LABGEO PT10, Samsung Electronics Co, Suwon, Korea). Repeatability and device/method precision of LABGEO.sup.PT10 was established according to the IVR-PT06 guideline. Serum insulin, leptin and adiponectin levels were measured with the Rat Insulin Kit (Linco Research Inc, St. Charles, Mo., USA) by ELISA (Elx-800; Bio-Tek Instruments Inc, Vermont, USA).
[0203] The total antioxidant capacity (TAC) was measured using an antioxidant assay kit (Sigma, St Louis, Mo., USA). Trolox was used as an antioxidant standard to calculate Trolox equivalent antioxidant capacity; absorbance readings were taken at 520 nm. Lipid peroxidation was measured in terms of MDA formation, which is the major product of membrane lipid peroxidation done by a previously described method (Karatepe, 2004) with slight modification.
[0204] The liver MDA content was measured by high performance liquid chromatography (HPLC, Shimadzu, Tokyo, Japan) using a Shimadzu UV-vis SPD-10 AVP detector and a CTO-10 AS VP column in a mobile phase consisting of 30 mM KH2PO4 and methanol (82.5+17.5, v/v; pH 3.6) at a flow rate of 1.2 ml/min. Column effluents were monitored at 250 nm and the volume was 20 l. The liver homogenate (10%, w/v) was prepared in 10 mM phosphate buffer (pH 7.4), centrifuged at 13,000g for 10 min at 4 C., and the supernatant was collected and stored at 80 C. for MDA analysis (Karatepe, 2004). Activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) of homogenized liver were measured using a commercial kit (Cayman Chemical, Ann Arbor, Mich., USA) according to the manufacturer's instructions.
[0205] Western Blots Analysis
[0206] For Western blot analyses protein extraction was performed by homogenizing the liver in 1 ml ice-cold hypotonic buffer A, containing 10 mM2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid [HEPES] (pH 7.8), 10 mMKCl, 2 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, and 0.1 mM phenylmethylsulfonyl-fluoride (PMSF). The homogenates were added with 80 l of 10% Nonidet P-40 (NP-40) solution and then centrifuged at 14,000g for 2 min. The precipitates were washed once with 500 l of buffer A plus 40 l of 10% NP-40, centrifuged, re-suspended in 200 l of buffer C [50 mM HEPES [pH 7.8], 50 mMKCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM dihiothreitol [DTT], 0.1 mM PMSF, 20% glycerol], and recentrifugedat 14,800g for 5 min. The supernatants were collected for determinations of NF-KB, VEGF, iNOS, ICAM, Nrf2, and HO-1 according to the method described by Sahin et al. [2012]. Equal amounts of protein (50 g) were electrophoresed and subsequently transferred onto a nitrocellulose membrane (Schleicher and Schuell Inc., Keene, N.H., USA).
[0207] Antibodies against NF-B, TNF-, Nrf2, HO-1, PPAR-, and p-IRS1, (Abcam (Cambridge, UK) were diluted (1:1000) in the same buffer containing 0.05% Tween-20. Protein loading was controlled sing a monoclonal mouse antibody against -actin (A5316; Sigma). Bands were analyzed densitometrically using an image analysis system (Image J; National Institute of Health, Bethesda, USA). (
[0208] Statistical Analysis
[0209] Sample size was calculated based on a power of 85% and a P-value of 0.05. Data are expressed as meanstandard deviation. Differences among the groups were evaluated using the General Linear Model (GLM) procedure of SAS at baseline. If ANOVA indicated significance, a Fisher's multiple comparison test was performed. The alpha level of significance was set at P<0.05.
[0210] Effect of Beta-Cryptoxanthin Compositions on Oxidative Metabolites and Antioxidant Capacity in Liver Tissue
[0211] Antioxidant capacity, catalase, superoxide dismutase (SOD) increased in BCX treated rats and decreased TBARS in liver. These results suggest BCX protects liver from oxidative stress due to hyperglycemia.
TABLE-US-00005 TABLE 5 ND HFD Details Control BC HFD BC Liver TBARS (nmol/mg protein) 1.82 1.56 3.82 2.98 Serum TAC (U/mL) 1.18 1.19 0.46 0.64 Liver SOD (U/mg protein) 182.86 198.57 106.29 130.43 Liver CAT 299.71 306.14 253.43 271.57 (U/mg protein) Liver GSHPx 41.29 50.29 16.86 17.43 (U/mg protein)
[0212] GSHPx: (Glutathione Peroxidase); CAT: (Catalase); SOD: (Superoxide Dismutase); TAC: (Total Antioxidant Capacity)
[0213] Rats fed a HFD had lower levels of total antioxidant capacity (TAC) activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and higher malondialdehyde (MDA) concentration than rats fed a standard diet (P<0.001 for all). Beta-cryptoxanthin administration increased activities of these enzymes and decreased MDA concentration in rats fed a HFD (P<0.05) (See
[0214] BCX compositions upregulate BCO2 expression. BCO2 acts as a protective antioxidant and plays a crucial role in protection against oxidative damage (see
[0215] It is also observed that decrease in TNF alpha decreases oxidative stress in liver tissue (see
[0216] BCX activated PPAR gamma in HFD treated rats shows that BCX had a significant role in CMS and antioxidant pathways (see
[0217] BCX compositions decrease Nrf2 expression, which improves glucose homeostasis, possibly through its effects on fibroblast growth factor 21 (Fgf 21) and/or insulin signaling in liver tissue of HFD rats (see
[0218] Further NFkB decreased in HFD treated with BCX. These results show BCX as antioxidant anti-inflammatory provitamin A (see
[0219] According to
[0220] Decreased IRS-1 was also associated with a decrease in glucokinase expression and a trend toward increased blood glucose. HFD rats treated BCX increased IRS-1 in liver so there is a potential decrease in blood glucose and glucose management (See
[0221] It is observed that beta-cryptoxanthin inhibited liver NFkB and TNF- expression by 22% and 14% and enhanced liver Nrf2, HO-1, PPAR-, and p-IRS1 levels were enhanced by 1.1.43, 1.41, 3.53, and 1.33, fold, respectively (P<0.001).